PHB is an energy storage material produced by a variety of bacteria in response to environmental stress and is a homopolymer of D-(-)-3-hydroxybutyrate which has properties comparable to polypropylene, Although poly-.beta.-hydroxybutyrate was first described over 60 years ago, the technological potential of the polymer and related polymers is only now being realized. There is increasing pressure to produce biodegradable plastics due to the problems of waste disposal in general, and disposal of long-lived plastics, specifically. Because PHB is biodegradable, there is considerable interest in using PHB for packaging purposes as opposed to other plastic materials in order to reduce the environmental impact of garbage. PHB also has utility in antibiotics, drug delivery, medical suture and bone replacement applications.
Despite PHB's advantages over other materials, its high cost of production has hindered its performance in the market. The production of the PHB polymer requires extensive treatment after the fermentation phase in order to purify the PHB. This treatment includes mechanical lysis, enzymatic treatment, detergent treatment, washing, agglomeration, and spray-drying. These procedures are expensive and also tend to break down the released granule of PHB.
Currently, two industrial entities are producing PHB. However, the costs of production and recovery of PHB are too high to ensure the use of the plastic as a commodity item. In the first case a regimen of enzymatic treatment and mechanical disruption is used to release the PHB granules from the cells, whereas in the second case, a chlorinated hydrocarbon is used to extract the PHB. In addition, "to the commercial methods currently employed" the related granules can be separated from cell debris by aggregation with salts, such as calcium chloride and other hardening agents, as described in the copending application Ser. No. 07/528,549, filed May 25, 1990.
In a copending patent application Ser. No. 07/362,514 filed Jun. 7, 1989, and in Slater et al., "Cloning and Expression in Escherichia coli of the Alcaligenes eutrophus H16 Poly-.beta.-Hydroxybutyrate Biosynthetic Pathway", J. Bact., 170:4431, (October 1988), E. coli was genetically transformed with genes from A. eutrophus which code for the PHB biosynthetic pathway. The cloning of the PHB pathway and its expression in E. coli were also later discussed in Schubert et al., J. Bact. 170:5837 (December 1988), Peoples et al., J. Biol. Chem. 264:15293 (1989a) and Peoples et al., J. Biol. Chem. 264:15298 (1989b). These patent applications and references and all references cited in this disclosure are expressly incorporated herein by reference.
Initial experiments indicated that PHB production in transformed E. coli has not been greater than about 50%, i.e., 30% (Schubert et al., supra.), 50% (Peoples et al., 1989b), 30-54% (Slater et al., supra.) of the bacterial cell weight. However, in a copending patent application to Dennis et al., Ser. No. 07/768,008, filed Sep. 27, 1991, improvements have been made to the transformed E. coli PHB production system that result in levels of intracellular PHB as high as 95% of the cell dry weight. PHB accumulation at this level results in a productivity in E. coli (grams produced per liter of culture per hour of time) which is significantly higher than the PHB productivity in A. eutrophus.
There is a need to develop a procedure wherein the PHB granules are gently and efficiently released from the E. coli cell. There is a further need to develop a PHB recovery system which is superior to the existing technology. The result of procedure would be more efficient production of PHB biodegradable plastic products at a greatly reduced cost.
It is therefore an object to the present invention to provide an improved method for producing PHB in transformed E. coli.
It is another object of this invention to provide an improved method for accumulating and recovering PHB from E. coli.
It is another object of this invention to provide transformed E. coli strains which produce high levels of PHB and which can be readily lysed to release such PHB.